Abstract
Tanshinones and phenolic acids are two major classes of bioactive compounds in Salvia miltiorrhiza. Revealing the regulatory mechanism of their biosynthesis is crucial for quality improvement of S. miltiorrhiza medicinal materials. Here we demonstrated that Smi-miR858a-Smi-miR858c, a miRNA family previously known to regulate flavonoid biosynthesis, also played critical regulatory roles in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza. Overexpression of Smi-miR858a in S. miltiorrhiza plants caused significant growth retardation and tanshinone and phenolic acid reduction. Computational prediction and degradome and RNA-seq analyses revealed that Smi-miR858a could directly cleave the transcripts of SmMYB6, SmMYB97, SmMYB111, and SmMYB112. Yeast one-hybrid and transient transcriptional activity assays showed that Smi-miR858a-regulated SmMYBs, such as SmMYB6 and SmMYB112, could activate the expression of SmPAL1 and SmTAT1 involved in phenolic acid biosynthesis and SmCPS1 and SmKSL1 associated with tanshinone biosynthesis. In addition to directly activating the genes involved in bioactive compound biosynthesis pathways, SmMYB6, SmMYB97, and SmMYB112 could also activate SmAOC2, SmAOS4, and SmJMT2 involved in the biosynthesis of methyl jasmonate, a significant elicitor of plant secondary metabolism. The results suggest the existence of dual signaling pathways for the regulation of Smi-miR858a in bioactive compound biosynthesis in S. miltiorrhiza.